The invention relates to agents and processes for detecting bacteria of the genus Listeria, in particular L. monocytogenes.
The members of the genus Listeria are gram-positive rod-shaped bacteria which occur ubiquitously. Seven different species belong to this genus: L. monocytogenes, L. ivanovii, L. seeligeri, L. welshimeri, L. innocua, L. murrayi and L. grayi. Of these, only L. monocytogenes is pathogenic for humans, endangering in particular the newly born, pregnant women and older persons, as well as patients subjected to immunosuppression. L. monocytogenes infections frequently have a fatal outcome.
Contaminated foodstuffs, in which the organism can multiply even at low temperatures around 4.degree. C., are frequently the cause of listeria infections. Thus, various listeria epidemics have been attributed to the consumption of contaminated food, such as, for example, raw milk, cheese or coleslaw. Consequently, rapid detection processes for detecting listerias, in particular in foodstuffs or clinical samples, are urgently required. These processes must additionally be able to distinguish between L. monocytogenes and the species which are not pathogenic for humans. Furthermore, it must be possible to detect all variants of L. monocytogenes, which is the species which is pathogenic for humans. Recent discussions resulted in the proposal to use L. innocua as indicator organism for a potential contamination with L. monocytogenes. Therefore, the detection of L. innocua would be very useful as well.
Detection of L. monocytogenes is effected in a known manner using processes which are based on culturing the microorganisms. The process described in Int. J. Food Microbiol. 4 (1987), 249-256 takes two weeks. A somewhat faster process is recommended by the International Dairy Foundation (IDF); however, it takes at least 6-8 days. Both processes are unsuitable for rapid identification because of the time they take. In addition, both processes are labor-intensive, since nutrient media must be inoculated repeatedly in order to obtain single colonies, and since the isolates must subsequently be characterized using biochemical and serological methods of investigation.
While the immunological tests which are currently on the market only take a few hours, they do not permit the important differentiation between different species of listerias. In these processes, also, a two-day pre-enrichment cultivation is required.
A method is described in Appl. Environ. Microbiol. 54 (1988), 2933-2937 in which L. monocytogenes is specifically detected using synthetic oligodeoxyribonucleotide probes. However, the probes which are used are not sufficiently specific, since they also react with the species L. seeligeri, which is not pathogenic for humans. Prior multiplication of the organisms is required for this process as well: samples of foodstuffs, or their dilutions, are spread on agar plates, and then the inoculated plates are incubated and subsequently investigated by the colony hybridization procedure using a radioactively labelled DNA probe. Detection takes place by autoradiography. This method, too, is labor-intensive and time-consuming.
The DNA sequence of the iap (invasion-associated protein) gene of L. monocytogenes is described in Infect. Immun. 58, 1943-1950 (1990). This gene encodes a protein which is also known under the designation p60 and which occurs in variants in all Listeria species. In L. monocytogenes this protein is responsible for the ability to invade animal cells. A polynucleotide (400 bases) having a component sequence from this gene is suitable as a DNA probe for distinguishing L. monocytogenes from other organisms.
The polymerase chain reaction (PCR) permits the in vitro amplification of nucleic acids, and prior cultivation is generally not necessary when using this process. In order to start the reaction, short nucleic acid fragments (primers) are required, which primers encompass the section of the genome which is to be amplified. Usually, two primers are required, each of which hybridizes with one nucleic acid strand. One of the primers therefore possesses the complementary sequence to the relevant section of the gene. The choice of these primers determines the specificity of the detection reaction. The use of this process for detecting L. monocytogenes is described in Appl. Environmental Microbiology 57, 606-609 (1991), in Letters Appl. Microbiol. 11, 158-162 (1990) and in J. Appl. Bact. 70, 372-379 (1991). More extensive information regarding the details of these processes is available in these publications. The DNA primers bind to the gene for listeriolysin, the listeria hemolysin. The specificity of these primers is at least uncertain as is evident from comments in J. Appl. Bact. 70: L. seeligeri cannot be differentiated with certainty from L. monocytogenes. The unambiguous detection of L. monocytogenes has thus hitherto not been possible using the PCR technique.
Polyclonal antibodies against L. monocytogenes p60 also react with the p60 protein of other, non-pathogenic Listeria species. Such antibodies are therefore unsuitable for specifically detecting L. monocytogenes by immunological processes. It is possible in principle to purify a polyvalent antiserum of this nature by the specific absorption of interfering antibody fractions: for this purpose, p60 protein from all the other Listeria species is covalently bound to carriers. The unwanted antibody fractions can be specifically absorbed; an antiserum then remains which only reacts with p60 protein from L. monocytogenes. This method for obtaining an L. monocytogenes-specific serum is elaborate: substantial quantities of the polyvalent antiserum are required as starting material, as are, in addition, the p60 iap gene products of the different Listeria species. The obtention of monoclonal antibodies against. p60 protein would not be associated with this large material requirement; nevertheless, the raising of antibodies against particular epitopes depends on chance: it is first of all necessary to prepare a large number of antibody-producing cell clones, from which suitable clones must then be selected. It has thus far not been possible to obtain antibodies in a targeted manner against epitopes which are specific for L. monocytogenes. The same holds true for epitopes which are specific for L. innocua.